Photoinduced Electron Transfer from the Tryptophan Triplet State in Zn-Azurin

Tryptophan is one of few residues that participates in biological electron transfer reactions. Upon substitution of the native Cu2+ center with Zn2+ in the blue-copper protein azurin, a long-lived tryptophan neutral radical can be photogenerated. We report the following quantum yield values for Zn-substituted azurin in the presence of the electron acceptor Cu(II)-azurin: formation of the tryptophan neutral radical (Φrad), electron transfer (ΦET), fluorescence (Φfluo), and phosphorescence (Φphos), as well as the efficiency of proton transfer of the cation radical (ΦPT). Increasing the concentration of the electron acceptor increased Φrad and ΦET values and decreased Φphos without affecting Φfluo. At all concentrations of the acceptor, the value of ΦPT was nearly unity. These observations indicate that the phosphorescent triplet state is the parent state of electron transfer and that nearly all electron transfer events lead to proton loss. Similar results regarding the parent state were obtained with a different electron acceptor, [Co(NH3)5Cl]2+; however, Stern–Volmer graphs revealed that [Co(NH3)5Cl]2+ was a more effective phosphorescence quencher (KSV = 230 000 M–1) compared to Cu(II)-azurin (KSV = 88 000 M–1). Competition experiments in the presence of both [Co(NH3)5Cl]2+ and Cu(II)-azurin suggested that [Co(NH3)5Cl]2+ is the preferred electron acceptor. Implications of these results in terms of quenching mechanisms are discussed.

Determination of quantum yields. NATA was used as the reference to determine the fluorescence and phosphorescence quantum yields for ZnAzW48. The excitation wavelength and bandpass were identical for NATA and ZnAzW48 spectra. The concentration of NATA was optimized such that the NATA absorbance was similar (within 3%) to the protein sample at the excitation wavelength. This optimization ensured that the protein and NATA samples experienced similar primary inner filter effects and the spectra could be analyzed with minimal additional corrections. Re-absorption of emitted light (secondary inner filter effect) was not taken into account because the sample absorbance at wavelengths above 310 nm is negligible.
The literature value of 0.13 for the fluorescence quantum yield of NATA 1 was used to calculate the fluorescence quantum yield (Φ ) and phosphorescence quantum yield (Φ ) of ZnAzW48. The analysis for determination of emission quantum yields was adapted from the literature 2 and modified to account for the presence of absorbing, but not emitting, moieties such as phenylalanine or the electron acceptor. The raw fluorescence spectrum was initially corrected by subtraction of the buffer-only spectrum from the raw spectrum; the integrated intensity of this corrected fluorescence spectrum for W48 is denoted and can be expressed as: A similar expression can be written for NATA: In both Eqs. (1) and (2) Collectively, the product of the first two terms on the right side of Eq. (1) is referred to as the light absorbed by W48 and the first term on the right side of Eq. The denominator is the rate of excitation of W48 in ZnAzW48, and this term has been described previously. 4 To determine Φ , the absorption feature at 515 nm from ZnAzW48• had to be isolated from the bleach at 628 nm because these regions partially overlapped. The overlap of the 628 nm and 515 nm features was corrected by adding a fraction of the pre-photolysis spectrum to the difference spectrum at a given timepoint such that the bleach of the 628 nm band was eliminated and the 650 to 800 nm region was flat. Because each difference spectrum exhibited a different amount of bleach, variable fractions of the pre-photolysis spectrum were needed to eliminate the bleach; these spectra in which the bleach was eliminated are referred to as corrected difference spectra (see below for an example). The intensities of the 515 nm peaks in the corrected difference spectra were plotted against total photolysis time to generate a single wavelength kinetic trace.
The literature value of 2200 M -1 cm -1 at 515 nm for • was used to convert the absorbance to concentration of ZnAzW48•. 4  In experiments with [Co(NH3)5Cl] 2+ or CuCl2 as the acceptor, there was no bleach at 628 nm and thus, corrected difference spectra did not need to be generated.
Above: Absorption and difference absorption spectra of a deoxygenated mixture of CuAzW48 and ZnAzW48 at a ratio ⁄ 0.80. Trace A is measured before photolysis and Trace B is collected 40 min after photolysis with 292 nm. Trace C is the difference spectrum calculated from -, the positive features at 515 and 488 nm are attributed to growth of the neutral radical while the negative feature centered at 628 nm shows the bleach of the Cu(II) absorbance band after reduction to Cu(I). Trace D is the corrected difference spectra calculated from where is the scalar value required to remove the negative feature at 628 nm. Different values of α are required for different time points. , and the normalized integrated intensity for NATA is , .
. The final normalized spectrum of NATA shown in Figure 1 is